APC precursors respond to appropriate cytokine signals by developing into functional APC capable of stimulating an antigen-specific immune response. Many pathways of tumor-specific immunity have been identified, but the most potent is that initiated by antigen-presenting dendritic cells (DC) and mediated by tumor-specific cytotoxic T-lymphocytes (Ostrand-Rosenberg et al 1994 Current Opinion in Immunology 6:722-727; Grabbe et al., 1995, Immunology Today 16:117).
Dendritic cells are a diverse group of morphologically and functionally similar APC present in small numbers in skin, liver, lung, spleen, blood, lymphoid organs, and bone marrow (Banchereau and Steinman 1998, Nature 392:245; Hart 1997, Blood 90:3245). The DC population is therefore complex, as it is comprised of cells at varying stages of maturation, as well as of cells derived from many different precursors (Banchereau and Steinman 1998, Nature 392:245; Hart 1997, Blood 90:3245).
In attempts to propagate and study DC in vitro, researchers have isolated either mature DC or their precursors from different sources including cord blood (Caux et al., 1996 J. Exp. Medicine 184:695-206), bone marrow (Ratta et al., 1998 Br J. Haematology 101:756-65), circulating CD34+ stem cells (Rosenzwajg et al., 1996, Blood 87:535), and peripheral blood monocytes (Kiertscher and Roth, 1996 J. Leukocyte Biol. 59:208-218), and exposed them to widely different culture conditions including human serum (Kiertscher and Roth, 1996 J. Leukocyte Biol. 59:208-218), fetal calf serum (Caux et al., 1996 J. Exp. Medicine 184:695-206), and various cytokines (reviewed in Hart 1997, Blood 90:3245), resulting in cells that exhibit a variety of different phenotypes and functions. One question that remains to be answered is how all of these cells are related and whether or not they should all be classified as DC.
In general, APC initiate tumor-specific immunity in vivo by the following pathway: immature APC take-up and process antigen, migrate to the lymphoid organs, and develop into mature APCs that present the processed antigen to T-cells, thereby stimulating an antigen-specific immune response.
The phenotype of APC precursors isolated from peripheral blood includes cells expressing the markers CD13, CD14, and CD33, suggesting that they are myeloid in origin (Kiertscher and Roth 1996 J Leukocyte Biol. 59: 208-218). In contrast, the phenotype of mature APC isolated from peripheral blood exhibit minimal CD14 while expressing high levels of HLA-DR (MHC class II), co-stimulatory molecules such as CD80, CD86 (June et al 1994 Immunol. Today; Young et al 1992 J. Clin. Invest. 90:229-237; Modino and Jenkins 1994 J. Leukocyte Biol. 55:805-815), and other cell surface molecules including CD4, CD11c, and CD83 (Kiertscher and Roth 1996 J. Leukocyte Biol. 59:208-218). Functionally mature APC can be propagated in vitro, in the presence of cytokines, from CD14+ precursor cells recovered from peripheral blood and from CD34+ cells recovered from cord blood, cytokine-mobilized adult blood, or bone marrow. APC that develop from precursors in vitro exhibit similar phenotypic and functional characteristics to those observed with circulating APC recovered from peripheral blood, including: high levels of expression of HLA-DR; expression of CD80, CD83, and CD86; and potent antigen-presentation activity capable of stimulating antigen-specific T-cells.
The Role of APC in Stimulating Anti-tumor Immunity in Animal Models
It has been postulated that APC are capable of inducing anti-tumor immunity, and that they are absolutely essential for this process to occur (Ostrand-Rosenberg et al 1994 Current Opinion in Immunology 6:722-727; Grabbe et al 1995 Immunology Today 16:117-120; Huang et al 1994 Science 264:961-965). Therefore, development of APC vaccines has been extensively investigated. In an early study, the role of APC in anti-tumor immunity was demonstrated by using as few as 2×105 primed DC to induce immunity when injected into naive mice (Inaba et al 1990 Intern. Rev. Immunol. 6:197-206). In a later study, the unique anti-tumor activity of DC was demonstrated when mouse DC pulsed with idiotype antigen from a B-cell lymphoma were injected into naive mice. The recipient mice were effectively protected from subsequent tumor challenges, and the treatment established a state of lasting immunity. Injection of antigen alone, or B-cells pulsed with antigen had no effect, suggesting that DC were responsible for the anti-tumor immunity (Flamand et al 1994 Eur. J. Immunol. 24:605-610). In a different study, mice were inoculated with a CD80 transfected tumor known to produce anti-tumor immunity, and only mice with MHC-compatible APC were capable of rejecting a subsequent tumor challenge (Huang 1994 Science 264:961-965). As a further test of this theory, it was demonstrated that bone marrow-derived DC can be pulsed with tumor-antigens in vitro then injected into syngeneic mice as an effective anti-tumor vaccine (Mayordomo et al 1995 Nature Medicine 1:1297-1302).
The Role of APC as stimulators of Anti-tumor Immunity in Humans
There is evidence that APC play a similar role in inducing anti-tumor immunity in humans. Peptide-specific cytotoxic T-lymphocytes were readily induced from purified CD8+ T-cells using peptide-pulsed DC, but were not induced when peptide-pulsed monocytes were used (Mehta-Damini et al 1994 J Immunology 153:996-1003). From a clinical perspective, four patients with advanced B-cell lymphomas were treated with DC that were recovered by density-gradient separation from their blood and pulsed with their own tumor idiotype antigen. This DC therapy resulted in a measurable reduction in the patient's B-cell lymphoma in three of the four treated patients (Hsu 1996, Nature Medicine 2:52). Similar results have been obtained using cytokine-induced DC, developed for ex vivo administration, from the peripheral blood of patients with prostate cancer and melanoma (Murphy 1996, Prostate 29:371; Nestle 1998, Nature Medicine 4:328). In both of these cases, the DC were pulsed with tumor-specific or tumor-associated antigens prior to being used as a vaccine, and reductions in tumor burden were observed in a minority of patients.
APC are Present at Low Levels in Cancer Patients
DC are limited in both number and function in human cancer patients. Immunohistological examination of renal cell cancer showed only 0.03% to 0.55% of the infiltrating leukocytes expressed the CD83 DC marker, and these cells failed to express important co-stimulatory molecules such as CD80 and CD86 (Troy et al 1998 Clin. Cancer Res. 4:585-593). In another study, purified CD83+ DC from both regressing and progressing melanoma metastases were examined for antigen-presenting activity. The DC from regressing metastases expressed CD86 and functioned as antigen-presenting cells, but the DC recovered from progressing metastases expressed little CD86 and induced T cell anergy instead of stimulation (Enk et al 1997 Internat. J. Cancer 73:309-316). Similarly, DC isolated from breast cancer patients were defective, and DC function inversely correlated with tumor stage (Gabrilovich et al 1997 Clin. Cancer Res. 3:483-490). Histopathology studies have long reported a correlation between the number of tumor-associated DC and patient survival (Furukawa et al 1985 Cancer 56:2651-2656; Schroder et al 1988 Am J. Clin. Path. 89:295-300; Ambe et al 1989 Cancer 63:496-503; Becker 1992 Anticancer Res. 12:511-520; Ishigami et al 1998 Oncology 55:65-69). Although the number of DC is low in cancer patients, large numbers of functional DC can be propagated in vitro in the presence of cytokines (Nestle et al 1998 Nature Med. 4:328-332; Murphy et al 1996 Prostate 29:371-380; Morse et al 1997 Ann. Surgery 226:6-16; Bernhard et al 1995 Cancer Res. 55:1099-1104). One important approach to cancer therapy is to stimulate viable APC precursors to develop into functional APC.
The Use of Cytokines in the Development of APC Immunotherapy
The development of DC immunotherapy for the treatment of human cancer has been a subject of intense study. However, several factor have limited DC research in the past, including DC rarity and the extensive immunoselection techniques required to recover them (Freundenthal and Steinman 1990 PNAS 87:7698; Thomas and Lipsky 1994 J. Immunol. 153:4016-4027; Kiertscher and Roth 1996 J. Leukocyte Biology 59:208-218). A major factor that has limited APC immunotherapy is the exceptionally rare frequency of mature APC in blood, where they constitute only 0.05% to 0.3% of the circulating mononuclear cells (Grabbe 1995 Immunology Today 16:117-120; Kiertscher and Roth et al 1996 J. Leukocyte Biology 59:208-218). However, new techniques now allow cytokines to be used in vitro to propagate large numbers of APC from peripheral blood (Romani et al 1994 J. Exp. Med 180:83-93). The combination of GM-CSF and IL-4 induces peripheral blood monocytes to differentiate into APC, resulting in a 100-fold amplification in the yield of dendritic APC in vitro (Kiertscher and Roth 1996 J. Leukocyte Biol. 59:208-218).
Recent immunotherapy methods have attempted to develop DC vaccines to treat human cancers. In several clinical trials, GM-CSF was used in combination with either IL-4 or TNFα to propagate DC in vitro from cells isolated from the patient's blood, the DC were loaded with tumor antigen of interest, then used to vaccinate patients with either B cell lymphoma (Hsu et al 1996 Nature Med. 2:52-58), melanoma (Nestle et al 1998 Nature Med. 4:328-332), or prostate cancer (Murphy et al 1996 Prostate 29:371-380). Anti-tumor immunity and objective clinical responses were observed in all of the studies. Although DC immunotherapy using ex vivo generated DC has been extensively studied, it may not be the most practical method to treat cancer patients. Blood must be drawn from the patient, manipulated in vitro to enrich the population of DC, and then administered back to the patient. This method presents the risk of contamination of the sample, and requires a facility dedicated to precursor cell enrichment and sterile manipulation of blood samples. This approach is limited by the amount of blood that can be safely drawn from the patient, or requires invasive leukophoresis techniques to extract precursor cells. In addition, little is known about the appropriate site or route of vaccination to optimize DC development, as intravenous and subcutaneous administration result in no detectable DC in the lymphoid organs or tumor site (Morse et al., 1999 Cancer Research 59:56-58).
GM-CSF and IL-4 Induces Differentiation of Potent APC in vitro
The combination of GM-CSF and IL-4 promotes differentiation of peripheral blood mononuclear cells (PBMC) into APC in vitro (Romani et al 1994 J. Exp. Med. 180:83-93; Sallusto et al 1994 J. Exp. Med. 179:1109-1118). More specifically, it has been demonstrated that CD14+ monocytes are the peripheral blood precursors that mature into DC that exhibit antigen-presenting activity (Kiertscher et al 1996 J. Leukocyte Biol. 59:208-218; Zhou and Tedder, 1996 PNAS 93:2588-92). GM-CSF is an independent trophic factor for both monocytes and DC (Markowicz et al 1990 J. Clin. Investigation 85:9955-961), while IL-4 primarily induces maturation along the DC developmental pathway (Peters et al 1993 Adv. Exp. Med. Biol. 329:275-280). GM-CSF provides the primary proliferative signal. In contrast, IL-4 induces myeloid precursors to decrease the expression of CD14 (Lauener et al 1990 Eur. J. Immunol. 20:2375-2381) with a concomitant increase in the expression of important antigen-presenting molecules, such as HLA-DR, CD80, CD86, and CD40 (Kiertscher and Roth; FIG. 1A). The effect of GM-CSF and IL-4 are synergistic, as the presence of both cytokines is required for in vitro transformation of DC precursor cell into mature APC (Sallusto et al 1994 J. Exp. Med. 179:1109-1118).
Antigen-presenting Phenotype and Activity is Dose and Time Dependent Upon the Combination of GM-CSF and IL-4: in vitro studies
The in vitro administration of GM-CSF in combination with IL-4 induced the development of APC from PBMC, in a dose and time-dependent manner. Adherent peripheral blood mononuclear cells were cultured in a fixed amount of GM-CSF and either no or increasing amounts of IL-4. The combination of GM-CSF and higher amounts of IL-4 are required to induce the development of APC having the following characteristic: an increase in the expression of important antigen-presenting molecules, such as HLA-DR, CD80, and CD86; a dose-dependent increase in endocytotic activity as measured by the capacity of the cells to take-up FITC-labeled dextran; and an increase in the capacity to present soluble antigens and stimulate antigen-dependent T cell responses. Thus, GM-CSF and IL-4 act synergistically to induce antigen-presenting activity that is up to 20 times greater than that observed with either cytokine alone.